Utility of Glycosylated TIMP3 molecules: Inhibition of MMPs and TACE to improve cardiac function in rat myocardial infarct model

Figure 1. Inhibitory activity of human MMP2, MMP9, and TACE. (A) Schematic diagrams of TIMP3 variant constructs. WT has an endogenous glycosylation site at Asn184 and labeled as CHO. Arrows indicate additional glycosylation sites. N‐TIMP3 was truncated in the C‐terminal domain and does not contain any glycosylation sites. (B and C) Inhibition dose response of human recombinant MMP2 an MMP9 for WT, K22S/F34N (v2), 5x glycosylated (v82) and N‐TIMP3. (D and E) Inhibition dose response of v82 fusion constructs (Fc, HSA, and Ab). (F) Inhibition dose response of human recombinant TACE for WT, K22S/F34N (TIMP3v2), 5x glycosylated (TIMP3v82), and N‐TIMP3. All reactions were for 90 minutes at 37°C incubation. Representative graphs are from n = 3 to 37 experiments. Data are means ± SD

Figure 2. TIMP3 activity in Zymography. (A) Recombinant human MMP9 was run on Gelatin Zymogram. TIMP3v2 (K22S/F34N) was incubated with Developing buffer overnight after cutting gel lanes and transferring into an individual container. After staining and destaining, the gel was quantified using ImageJ software (lower graph). The gel is a representative of n = 3 independent experiments (mean ± SEM). (B) TIMP3v2 (K22S/F34N) inhibition of cardiac tissue MMP activity on the Zymogram from pig cardiac lysates (50 μg/lane) after 4.5 hours post‐MI. Cardiac tissue was homogenized in RIPA buffer with PMSF. After staining and destaining, the gel was quantified using ImageJ software (lower graph). The gel is a representative of n = 3 experiments (mean ± SEM). Graphs are averages of each ImageJ scan. *P < 0.05 (vs control), **P < 0.01 (vs control)

Figure 3. Cardiac and plasma half‐life of TIMP3. (A) Alexa Fluo 680‐labeled TIMP3 (AF680‐K22S/F34N TIMP3, v2, 2 mg/rat) and (B) AF680‐N‐TIMP3 (1 mg/rat) were delivered directly into rat myocardium after 3 hours MI, and hearts were harvested at desired time points postinjection (n = 3 per time point). *P < 0.05 (vs control), **P < 0.01 (vs control). (C and D) Rat plasma half‐life of K22S/F34N TIMP3 (C) and N‐TIMP3 (D) after 6 hours continuous jugular vein infusion in 3 mg/kg/h and 10 mg/kg/h before collecting blood for 4 hours postdose (n = 3 per dose). Data are means ± SEM, *P < 0.05 (vs control), LLOQ, lower limit of quantitation

Figure 4. Echocardiography of LAD ligated MI rats. (A‐C) Intramyocardial injection; (A) N‐TIMP3 (1 mg/rat) was injected to directly to the myocardium via 31G needle after 3 hours LAD ligation in SD rats (n = 8). Echocardiography was performed by long axis scanning at Day 3 and Day 7 post‐MI condition. (B) TIMP3v2 (2 mg/rat) was injected directly to the myocardium after 3 hours LAD ligation in SD rats before echocardiography at Day 3 and Day 7 postinjection (n = 8‐10). (C) TIMP3v82 (2 mg/rat) was delivered via tail vein injection after 3 hours LAD ligated MI rats before echocardiography at Day 3 and Day 7 postinjection (n = 8). *P < 0.05 (vs control), two‐tailed t test. (D) No difference was observed with longer lasting TIMP3v82‐Fc infusion. v82‐Fc (5 mg/kg, single IV bolus injection) was infused via tail vein injection post‐LAD ligation (3 hours) in SD rats before echo scanning (n = 8‐9 per group) at Day 7. Data are mean ± SEM

Figure 5. Binding capacity of TIMP3 to acellular ECM. (A,B) TIMP3 molecules were incubated with ECM in 96‐well and washed with PBS and incubated with C‐terminal anti‐TIMP3 antibody (A) and N‐terminal anti‐TIMP3 antibody (B), and the bound protein was detected by IR800‐conjugated secondary antibodies and near IR imaging. After repeated washing with PBS, bound TIMP3 in duplicate wells was determined by near‐IR Odyssey LiCor image. (C,D) No wash includes without wash/fixed and aspirate (duplicates per wash condition). (C) The average of signal intensity for each treatment condition (n = 3). Data are means ± SEM, *P < 0.05 (vs no wash control), paired t test

Figure 6. Binding kinetics of TIMP3. (A) TIMP3v2 was incubated with ECM and washed with PBS. ECM bound TIMP3 was stained with anti‐TIMP3 Ab and bound protein was detected by IR800 conjugated secondary antibody and near IR imaging (duplicates wells). (B) Time course of IR800‐labeled TIMP3v2 (1 mg/kg, tail vein) accumulation into the left ventricular infarct area in rat MI hearts (3‐week post‐MI, n = 3 per time point). *P < 0.05 (vs control), paired t test. (C) Plasma levels of IR800‐TIMP3v2 were detected in SDS‐PAGE gel at 0.5, 1.5, 3, and 10 minutes postinjection

Figure 7. TIMP3 inhibition of collagen fibril degradation by collagenase: Collagenase type‐I (100 μg/well) was incubated with acellular ECM in the presence of varying concentrations of TIMP3v2 (0, 1 μg, 3 μg, 10 μg, 30 μg, 100 μg), N‐TIMP3 (300 μg) or MMP inhibitor Marimastat (100 μmol/L) for 1 hour at 37°C with gentle shake before fixing with 4% PFA. Collagen fibril degradation was assessed by immunostaining using anti‐Collagen type III antibody. Scale bar = 200 μm. Graph is the average of n = 3 experiments with SEM. *P < 0.05 (vs control), **P < 0.01 (vs control), paired t test

Figure 8. Bioactivity of ECM bound TIMP3. Varying concentrations of TIMP3 were incubated with ECM and washed repeatedly with PBS to remove unbound. Inhibition of MMP2 and MMP9 enzyme activity was assessed by FRET‐based assay for the ECM bound TIMP3v2 (A and B) and N‐TIMP3 (C and D). Graphs are the average of n = 3 experiments (mean ± SEM), *P < 0.05 (vs control), paired t test

Figure 9. Working hypothesis of TIMP3 in the acute heart failure. MMP and TIMP3 levels are balanced in normal condition. In normal condition, cardiomyocytes (CM) and cardiac fibroblasts (CF) are spatially well organized in the myocardium. Upon ischemic insults, loss of cardiomyocytes is followed by ECM degradation by elevated MMPs. Eventually, myofibroblasts will trigger fibrosis in the infarcted area that leads to adverse remodel in the heart